Our studies of the tryptophan synthase alpha-2-beta-2 complex are aimed at relating structure to function. The tryptophan synthase multienzyme complex is an excellent model system for investigating enzyme mechanism, protein-protein interaction, metabolite channeling, and ligand-dependent site-site interactions. We are continuing to correlate structural information obtained by x-ray crystallography of the tryptophan synthase alpha-2-beta-2 complex from Salmonella typhimurium with information obtained on enzyme mechanism and on protein folding by other techniques. We are also using the crystal structure to select residues for further exploration by site-directed mutagenesis. Our studies with mutants are clarifying the mechanism of the reactions catalyzed by the beta subunit. We find that certain residues play catalytic roles whereas other residues control the reaction specificity or substrate specificity of the enzyme. Spectroscopic studies demonstrate that chromophoric intermediates are formed between pyridoxal phosphate and substrates at the active site of the beta subunit both in solution and in the crystalline state. We find that the equilibrium distribution of intermediates is affected by pH, by ligands of the beta subunit, and by mutation. Rapid-scanning stopped-flow spectroscopic studies are used to measure the rates of formation of inter- mediates in the reactions of wild type and mutant forms of tryptophan synthase The results shed light on the mechanisms of catalysis, of ligand-dependent site-site interactions, and of metabolite channeling. Scanning microcalorimetric and circular dichroism studies of the thermal unfolding of wild type and mutant forms of the a subunit demonstrate features that are responsible for protein stability. Rapid quench-flow studies under single-turnover conditions provide evidence for the existence of a channeled indole intermediate.